1 Improving seed germination of the eggplant rootstock Solanum torvum by testing multiple
2 factors using an orthogonal array design
3
4 R.H.G. Ranila, H.M.L. Niranb, M. Plazasc, R.M. Fonsekaa, H.H. Fonsekad, S. Vilanovac, I. Andújarc,
5 P. Gramazioc, A. Fitac, J. Prohensc,*
6
7 aDepartment of Crop Science, Faculty of Agriculture, University of Peradeniya, Old Galaha Road,
8 Peradeniya 20400, Sri Lanka
9 bAgriculture Research Station, Girandurukotte 90750, Sri Lanka
10 cInstituto de Conservacion y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de
11 València, Camino de Vera 14, 46022 Valencia, Spain
12 dHorticultural Crop Research and Development Institute, Gannoruwa Road, Peradeniya 20400, Sri
13 Lanka
14
15 *Corresponding author. Tel: +34 963879424; fax: +34 963879422.
16 E-mail addresses: [email protected] (R.H.G. Ranil), [email protected] (H.M.L. Niran),
17 [email protected] (M. Plazas), [email protected] (R.M. Fonseka),
18 [email protected] (H.H. Fonseka), [email protected] (S. Vilanova),
19 [email protected] (I. Andújar), [email protected] (P. Gramazio), [email protected] (A. Fita),
20 [email protected] (J. Prohens)
21
22
1
23 ABSTRACT
24 Solanum torvum is a highly vigorous relative of eggplant that is resistant to a number of harmful
25 soil-borne diseases and is compatible for grafting with eggplant. Being a potential rootstock, this
26 plant frequently presents poor and erratic germination, which makes its practical use difficult. We
27 used an L8 (27) orthogonal array design to evaluate the primary effects of seven factors (soaking of
28 seeds, scarification with sodium hypochlorite (NaClO), application of gibberellic acid (GA3), use of
29 potassium mitrate (KNO3) as a moistening agent, cold stratification, application of a heat shock, and
30 light irradiation during germination) at two levels (L0 and L1) using four germination parameters
31 (early and final germination, germination rate and vigour index) in fresh S. torvum seeds. Solanum
32 torvum seeds had a strong dormancy with no germination in the untreated seeds and high early and
33 final germination (approximately 100%) in certain treatments. An evaluation of the main effects
34 revealed highly positive effects on germination from seed soaking, and the use of GA3, KNO3, and
35 light irradiation, whereas NaClO scarification had a negative effect. The application of cold
36 stratification and heat shock treatments also had a positive effect on seed germination but to a lesser
37 extent than the other treatments. An improved proposed protocol that consisted of subjecting seeds
38 to soaking, the application of GA3 and KNO3, cold stratification, heat shock, and light irradiation
39 was validated and demonstrated to be highly effective, with seed germination success greater than
40 60% being observed at 3 d and final germination reaching a plateau at 6 d. A second validation
41 experiment using a commercial growing substrate also showed a high emergence (approximately
42 50%) at 7 d and a final germination of approximately 80% was recorded with application of the
43 improved protocol. The seed germination protocol that we have developed will facilitate the use of
44 S. torvum as a rootstock for eggplant and its use in breeding programmes. Our results also reveal
45 that orthogonal array designs are a powerful tool for establishing improved protocols for seed
46 germination.
47
48 Keywords: Solanum torvum, seed, germination, dormancy, orthogonal arrays, main effects
2
49
50 1. Introduction
51
52 Solanum torvum Sw., commonly known as turkey berry, devil’s fig or pea eggplant, is a
53 wild bush of neotropical origin that belongs to the “spiny Solanum” (subgenus Leptostemonum)
54 group (Levin et al., 2006). This species has become naturalised and is sometimes invasive in
55 tropical areas of Africa, Asia, and Australia; also, this species is occasionally cultivated, primarily
56 in Southeast Asia and Africa, for its edible fruits (Gousset et al., 2005; Nyadanu and Lowor, 2015).
57 Solanum torvum is of great interest as a rootstock for eggplant (S. melongena L.), as the plant is
58 highly vigorous, fully graft-compatible with eggplant scions (Gisbert et al., 2011b; Moncada et al.,
59 2013), and possesses resistance to a wide range of soil pathogens, such as Verticillium dahliae,
60 Ralstonia solanacearum, Fusarium oxysporum, and root-knot-nematodes (Bletsos et al., 2003;
61 Gousset et al., 2005; Bagnaresi et al., 2013), as well as being tolerant to abiotic stresses (Schwarz et
62 al., 2010). Furthermore, eggplant fruits produced on scions grafted onto S. torvum are of good
63 quality (Gisbert et al., 2011b; Moncada et al., 2013; Miceli et al., 2014). Additionally, grafting
64 eggplant on S. torvum reduces translocation of the heavy metal cadmium (Cd) from the roots to the
65 aerial part (Arao et al., 2008) and may minimize the negative effects on the fruit quality from Cd
66 soil contamination (Savvas et al., 2010). Because of these desirable traits, S. torvum also represents
67 a genetic resource of strong relevance to the introgression breeding of eggplant (Kumchai et al.,
68 2013)
69 The primary limitation for the practical use of S. torvum as a rootstock in the commercial
70 production of grafted eggplant plants, as well as in breeding programmes, is the poor, irregular and
71 erratic germination due to dormancy in seeds (Ginoux and Laterrot, 1991; Miura et al., 1993;
72 Gousset et al., 2005; Hayati et al., 2005). This characteristic has even led to the proposed use of
73 vegetative propagation to overcome the seed germination problem (Miceli et al., 2014). The
74 breaking of dormancy, which is a common phenomenon among wild Solanum species (Taab and
3
75 Andersson, 2009; Wei et al., 2010; Kandari et al., 2011; Tellier et al., 2011), and the enhancement
76 of germination can be achieved using combinations of many different physical (e.g., seed soaking,
77 manual scarification, cold stratification, heat shocks, light irradiation, and magnetic fields) and/or
78 chemical (e.g., scarification with acidic or basic chemicals, plant growth regulators, and osmotic
79 treatments) treatments (Finch-Savage and Leubner-Metzger, 2006; Bewley et al., 2013; Holubowicz
80 et al., 2014).
81 Determining the critical combination of factors that permit the enhancement of germination
82 in S. torvum seeds is important to develop improved protocols for seed germination in this species
83 for its use as a rootstock and for breeding purposes (Hayati et al., 2005; Gisbert et al., 2011a). The
84 influence of several potentially key factors affecting a variable, in this case seed germination, can
85 be determined by studying one factor at a time (as achieved by Hayati et al., 2005). However, this
86 process greatly reduces efficiency when there is an interdependency of factors or when it is
87 impractical to isolate and test each variable individually. Full factorial designs, which are much
88 more efficient in determining the optimal combination of factors, may require large and costly
89 experiments when many factors are involved (Onyiah, 2008; Rao et al., 2008). An alternative
90 commonly used in industrial applications are orthogonal (Taguchi) arrays (Roy, 2010), which allow
91 the main effects of a large number of factors to be estimated with a limited number of treatments.
92 Although orthogonal arrays have been successfully applied to address the problem of determining
93 adequate combinations of factors in biological and biotechnological processes (Rao et al., 2008;
94 Assemi et al., 2012; Sedghi et al., 2014; Vasilev et al., 2014), their use for establishing seed
95 germination protocols has been highly limited (Wu et al., 2011; Poinapen et al., 2013) and largely
96 overlooked.
97 In this work, we evaluate the primary effects of seven factors potentially involved in the
98 release from dormancy and enhancement of seed germination in dormant seeds of S. torvum using
99 an orthogonal array experimental design. The improved protocol established according to the results
100 was then tested and validated. The results from this work will provide information for improving
4
101 the seed germination of S. torvum. These findings will also contribute to facilitating its use as a
102 rootstock and as a source of variation in breeding programmes. At the same time, this work aims to
103 demonstrate the potential of orthogonal array designs for establishing efficient seed germination
104 protocols.
105
106 2. Materials and Methods
107
108 2.1. Seed materials and germination conditions
109
110 Fresh seeds of S. torvum accession No. 55953 (originally purchased from Sunshine Seeds,
111 Ahlen, Germany) were extracted from physiologically ripe fruits of plants cultivated in an open
112 field at Universitat Politècnica de València (Valencia, Spain). Five-month-old seeds of S.
113 melongena accession No. BBS-188/B (landrace from Ivory Coast), with high germination values
114 (>90%), were also used as a control for the validation of the improved treatment developed for the
115 germination of S. torvum seeds.
116 Depending on the experiment, seeds were germinated in Petri dishes (9.0 × 2.5 cm; Phoenix
117 Biomedical, Mississauga, Ontario, Canada) on a layer of 0.5 cm of embedded hydrophilic cotton
118 covered by filter paper or sown at a depth of 7 mm in plastic pots (9 × 9 × 9.5 cm) containing
119 commercial nursery growing substrate (Neuhaus Huminsubstrat N3, Lassmann-Dellmann, Geeste,
120 Germany). Twenty-five evenly distributed seeds were placed in each Petri dish or pot. Seeds were
121 sown in Petri dishes and pots at the beginning of the experiments (day 0) in a climatic chamber with
122 a 14-h light / 10-h dark photoperiod at 25ºC constant temperature. A light irradiance of 600
123 mmol·m-2·s-1 was provided by GRO-LUX F36W/GRO (Sylvania, Danvers, MA, USA) fluorescent
124 tubes. The pots were watered regularly to keep the substrate moistened.
125
126 2.2. Factors evaluated
5
127
128 Seven factors, soaking, sodium hypochlorite (NaClO), gibberellic acid (GA3), potassium
129 nitrate (KNO3), cold, heat, and light, with two possible levels (level 0, L0; level 1, L1) for each
130 factor were evaluated for their effects on the germination of S. torvum seeds. The levels for each
131 factor were as follows:
132 (a) Soaking: L0 = no soaking; L1 = soaking seeds in water for 1 d.
133 (b) NaClO: L0 = no NaClO scarification; L1 = NaClO scarification by the immersion of
134 seeds for 10 min in a 1.2% NaClO (SPB, Cheste, Spain) solution followed by the rinsing of seeds
135 with water.
136 (c) GA3: L0 = no GA3 application; L1 = soaking seeds in a 500 ppm solution of GA3
137 (Duchefa Biochemie, Haarlem, The Netherlands) for 1 d.
138 (d) KNO3: L0 = use of water as a moistening agent (when using germination in Petri dishes)
139 or for watering (when using germination in growing substrate); L1 = use of a 1,000 ppm KNO3
140 (Panreac, Montcada i Reixac, Spain) solution as a moistening agent or as a watering solution.
141 (e) Cold; L0 = no cold stratification; L1 = seed stratification by placing moist seeds already
142 deposited on Petri dishes with a moistening agent or sown in seedling trays within a wet nursery
143 growing substrate at 4ºC for 7 d.
144 (f) Heat; L0 = no heat shock; L1 = placing moist seeds already deposited on Petri dishes
145 with a moistening agent or sown in seedling trays within a wet nursery growing substrate at 37ºC
146 for 1 d.
147 (g) Light: L0 = seeds placed in darkness (Petri dishes covered with aluminium foil); L1 =
148 seeds subjected to light irradiation (16 h of light at an intensity of 600 mmol·m-2·s-1 / 8 h dark).
149 The light factor was considered only for experiments involving the evaluation of
150 germination in Petri dishes. For the experiment involving sowing seeds in a commercial substrate,
151 all seeds were covered with a 7-mm layer of substrate.
6
152 The factors soaking, NaClO and GA3 were applied before sowing seeds on Petri dishes or in
153 the nursery growing substrate. The factors KNO3, cold and heat were applied after sowing seeds,
154 but before initiation of the evaluation of germination or emergence (day 0). The light factor was
155 applied at the initiation of the experiment (day 0). Factors were applied one after the other
156 according to the following order: (1) soaking, (2) NaClO, (3) GA3, (4) KNO3, (5) cold, (6) heat, and
157 (7) light. As L1 levels for some of the factors involve pre-germination procedures that may last up
158 to 7 days, their application was programmed so that the initiation of the evaluation of germination
159 or emergence (day 0) was synchronized for all treatments of a given experiment (Table 1).
160
161 2.3. Traits evaluated
162
163 Seed germination was evaluated at 0, 4, 6, 8, 11, 13 and 15 d after the seeds were placed in
164 the germination cabinet (day 0) for the first experiment (Petri dishes germination), which was aimed
165 at determining the levels of different factors for improving the germination of S. torvum. For the
166 experiments aimed at validating the improved treatment, the seed germination (Petri dishes) or
167 emergence (growing substrate) was evaluated at 0, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13 and 14 d after the
168 seeds were placed in the germination cabinet (day 0). Seeds were considered germinated when the
169 radicle was 1 mm or greater. Emergence was evaluated by counting germinating seedlings.
170 The following four parameters were considered for an analysis of variance (ANOVA)
171 statistical evaluation (Ranal and Garcia de Santana, 2006): (a) early germination/emergence
172 (measured at 4 d or 5 d, depending on the germination experiment, and at 7 d for emergence; %);
173 (b) final germination/emergence (measured at 14 d or 15 d, depending on the experiment; %); (c)
174 germination/emergence rate, which determines the potential for a high final germination combined
175 with a rapid germination/emergence, calculated as (S1×t1 + S2×t2 + … + Sn×tn) / (t1 + t2 + … + tn),
176 where Sn is the cumulative percentage of germinated seeds at germination test n and tn is the
177 number of days at which test n was performed, expressed as a percentage (%); and, (d) vigour
7
178 index, which determines the potential for a rapid germination/emergence, calculated as
179 (S1/t1)+(S2/t2)+ … +(Sn/tn).
180
181 2.4. Establishing an improved seed germination treatment
182
7 183 The main effects of the seven factors studied at two levels were evaluated using an L8 (2 )
184 orthogonal array design (Roy, 2010) consisting of eight treatments (Table 1). These eight treatments
185 are orthogonal and each of the two levels (L0 and L1) for each factor is represented in the different
186 treatments the same number of times (four), of which for any factor one half (two) are evaluated at
187 level L0, and the other half (two) are evaluated at level L1 for any other factor. For each treatment,
188 six replicates (six Petri dishes, with 25 seeds per Petri dish) were used. Data on the four studied
189 parameters were transformed using the arcsine transformation (inverse sine of the square root of
190 percentage/100 for percentage data, and the proportion of the maximum possible value for the
191 vigour index) and subjected to an ANOVA for testing the significance of differences of the
192 treatments (Little and Hills, 1978). The significance of differences among the treatment means of
193 transformed data was evaluated in transformed data using the Student-Newman-Keuls multiple
194 range test at a P=0.05 (Hsu, 1996).
195 The degrees of freedom and sums of squares of the ANOVA for the eight treatments were
196 partitioned in seven orthogonal contrasts for testing the significance of the main effect (i.e., the
197 difference in the average between levels L0 and L1) for each factor (Little and Hills, 1978). Using
198 this information, we proposed an improved protocol for the germination of S. torvum by including
199 the level of each factor having a positive significant effect on the seed germination parameters
200 studied.
201
202 2.5. Validation of the improved germination treatment
203
8
204 A first experiment was performed to evaluate if the orthogonal array method had been
205 efficient in establishing an improved germination protocol under the experimental conditions (Petri
206 dish germination) used for developing it. To validate the method used to establish the improved
207 treatment we compared two treatments: (a) the proposed improved germination treatment according
208 to the results of the orthogonal array experiment and (b) the best treatment out of the eight tested in
209 the orthogonal array matrix. Six replicates (six Petri dishes, with 25 seeds per Petri dish) for each of
210 these two treatments were used. Analyses and significance of differences of transformed data were
211 performed by using an ANOVA as mentioned in section 2.4.
212 A second experiment was conducted to evaluate if the improved germination treatment
213 obtained with the orthogonal array method under experimental conditions (germination in Petri
214 dishes) was useful in improving the germination of S. torvum under commercial nursery conditions,
215 by evaluating the emergence of seeds sown in a nursery growing substrate. In this experiment, the
216 light factor had to be set at the level L0, as seeds were sown at a depth of 7 mm and germinated in
217 the dark in all cases. The three treatments evaluated were a) Solanum torvum control treatment (all
218 factors at level L0), b) the proposed improved germination treatment according to the results of the
219 orthogonal array experiment, and c) Solanum melongena control treatment (all factors at level L0).
220 Six replicates (six pots) for each of these three treatments were used. Data analyses and significance
221 of differences were performed by using an ANOVA with transformed data as mentioned in section
222 2.4.
223
224 3. Results
225
226 3.1. Establishing an improved seed germination treatment
227
228 Highly significant differences (P<0.0001) were observed in the ANOVA analysis among the
229 eight treatments (1 to 8) evaluated in the orthogonal array design for the four traits evaluated (Table
9
230 2). Treatments 5 and 6, which share levels L1 for soaking, L0 for NaClO and L1 for GA3 (Table 1),
231 had a high early germination, being significantly superior to the rest of the treatments (Figure 1).
232 For treatments 2, 3, and 4 an early germination of some seeds was observed, but no significant
233 differences were observed among them. The remaining treatments (1, 7, and 8) presented no
234 germination at 4 d (Figure 1). The highest final germination at the end of the experiment (15 d) was
235 found for treatments 2, 5 and 6 with average germination values above 99% and significantly higher
236 values than the other treatments (Figure 1). Intermediate germination values were recorded for
237 treatments 3, 4 and 8, with treatments 3 and 8 presenting significantly higher values than treatment
238 4. Finally, treatment 7 had a very low germination and treatment 1 no germination at all.
239 Observation of the seeds sown in Petri dishes applying treatments 1 and 7 did not reveal any further
240 germination even after 1 month.
241 The germination rate was highest (>98%) in treatments 5 and 6, which was significantly
242 higher than that of the rest of the treatments (Figure 1). The next treatment with the highest
243 germination rate was treatment 2, which was significantly higher than that of the treatments 3, 8 and
244 4. The lowest germination rate values were obtained for treatments 1 and 7 (Figure 1). The vigour
245 index followed a similar pattern as the germination rate, with the highest values being those in
246 treatments 5 and 6, and the lowest values coinciding with treatments 1 and 7. The significant groups
247 for the vigour index were identical to those observed for the germination rate (Figure 1).
248 The orthogonal contrasts obtained from the partition of the degrees of freedom and sums of
249 squares of the ANOVA for the treatments revealed that significant differences existed among the
250 average values for the two levels (L0 and L1) for all factors in the four parameters studied, with the
251 exception of the cold factor for the germination rate and vigour index (Table 2). The greatest F-
252 ratios (P<0.0001) for early germination were obtained for the orthogonal contrasts of GA3, NaClO
253 and soaking. The remaining orthogonal contrasts were significant at P<0.01. For the final
254 germination, all orthogonal contrasts were highly significant (P<0.0001) except for the cold factor
255 (P<0.01), with the highest values being those for light, KNO3, NaClO and GA3. For the germination
10
256 rate, all the orthogonal contrasts were highly significant (P<0.0001), except for cold (Table 2). The
257 highest F-ratio values were obtained for GA3, light, KNO3, and NaClO. For the vigour index, again
258 cold was non-significant, and the remaining orthogonal contrasts were highly significant
259 (P<0.0001), except for heat, which was significant at P<0.01 (Table 2).
260 The average values of level 1 (L1) were greater than those of level 0 (L0) for all factors
261 across the four parameters studied with the exception of the NaClO factor, in which the values were
262 greater for L0 (Table 3). For early germination, the highest L1-L0 absolute differences between
263 levels were for GA3, NaClO, and soaking, with values ≥40.0%. For the remaining factors these
264 differences were <5% (Table 3). In the case of late germination and germination rate, the greatest
265 differences between L1 and L0 were for the light, KNO3, NaClO, and GA factors. Finally, for the
266 vigour index, the greatest absolute differences were found for factors GA3, NaClO, soaking, and
267 light (Table 3).
268 Based on the results obtained from the orthogonal contrasts for the primary effects of each
269 factor tested and the average values for each of the levels of each factor, the following improved
270 germination treatment is proposed for enhancing S. torvum seed germination: soaking: L1, NaClO:
271 L0, GA3: L1, KNO3: L1, cold: L1, heat: L1, and light: L1 (Figure 1).
272
273 3.2. Validation of the improved germination treatment
274
275 The improved germination protocol proposed in section 3.1 was not among the treatments
276 tested in the orthogonal array design. To validate the proposed treatment for its germination in Petri
277 dishes, we compared it with treatment 6 of the orthogonal array design. Treatment 6, together with
278 treatment 5, was significantly superior to the other treatments for all the parameters studied (except
279 for the final germination of treatment 2, which did not differ significantly from treatments 5 and 6).
280 Treatment 6 was chosen over treatment 5, as the former had a slightly higher (although non-
281 significant) early germination (Figure 2). The improved treatment and treatment 6 differ in cold (L1
11
282 for the improved treatment and L0 for treatment 6) and light (L1 for the improved treatment and L0
283 for treatment 6) factors, with the remaining treatments applied at the same levels. No significant
284 differences were obtained between the improved treatment and treatment 6 for any of the
285 germination parameters studied (Table 4). The germination curves for both treatments are very
286 similar, although values for the improved treatment are higher (although not significantly different)
287 than those of treatment 6 (Figure 3). Germination occurred very quickly with more than 60% of the
288 seeds germinated at 3 d and a germination plateau achieved at 6 d (Figure 3).
289 Regarding validation of the proposed method in the nursery growing substrate, highly
290 significant differences (P<0.0001) were observed among the three treatments tested (S. torvum
291 control, S. torvum improved treatment, and S. melongena control) for the seed emergence traits
292 evaluated (Table 4). The seeds of S. torvum with the control treatment did not germinate (Table 5).
293 Solanum torvum seeds with the improved treatment had approximately 50% early emergence (at
294 day 7), which is significantly higher than that of the S. melongena control (Table 5). Conversely, the
295 final germination of the control seeds of S. melongena was significantly higher than that of the S.
296 torvum improved treatment. This resulted in a sharper sigmoidal curve in the S. melongena control
297 compared to the S. torvum improved treatment (Figure 4). Regarding the emergence rate, no
298 significant differences were observed between the S. melongena control and the S. torvum improved
299 protocol (Table 5). However, for the vigour index, the values for the S. torvum improved treatment
300 were significantly higher than those of the S. melongena control (Table 5). For both treatments,
301 germination reached a plateau in 10 d.
302
303 4. Discussion
304
305 Although S. torvum is considered to be an outstanding rootstock for the commercial
306 production of eggplant (Miceli et al., 2013; Moncada et al., 2014), its practical utilization is
307 hampered by dormancy and poor germination (Ginoux and Laterrot, 1991; Miura et al., 1993;
12
308 Hayati et al., 2005). The efficient and successful production of high-quality grafted vegetable plants
309 requires an adequate synchronization of the development of rootstock and scion plantlets, which
310 requires the predictable germination of both the rootstock and scion (Lee et al., 2010). In this
311 respect, the germination protocol described here, which involves the application of a combination of
312 different factors having a positive effect on the germination of dormant seeds of S. torvum, has
313 proved highly efficient in producing a reliable, rapid and uniform germination in this species.
314 The use of an L8 orthogonal array experimental design allows the main effects on S. torvum
315 seed germination to be determined for seven factors using only eight treatments in which factors are
316 arranged in an orthogonal matrix (Roy, 2010). A few studies use orthogonal arrays for improving
317 seed germination in other species by studying only three (Wu et al., 2011) or four factors (Poinapen
318 et al., 2013). To the best of our knowledge, the present study is conducted with the largest number
319 of factors evaluated for improving seed germination. Our results show that similar to industrial and
320 biotechnological processes (Rao et al., 2008; Roy, 2010), orthogonal arrays are robust, powerful
321 and simple tools for simultaneously studying the primary effects of a large number of factors to
322 improve seed germination protocols in horticultural species. The primary advantages of orthogonal
323 arrays for seed germination testing is that they are much more efficient than studying one variable at
324 a time, and they are much simpler and less costly than full factorial designs (Little and Hills, 1978;
325 Onyiah, 2008; Rao et al., 2008).
326 All factors included in this study are known to have a potential effect on seed germination
327 (Finch-Savage and Leubner-Metzger, 2006; Bewley et al., 2013). In the present orthogonal array
328 experiment, we observed that all of the factors had an effect, although of varying aspects and
329 magnitude, on the seed germination of S. torvum seeds. The factors that exhibited a larger effect on
330 different seed germination parameters studied were soaking, NaClO, GA3, KNO3 and light. In all
331 treatments, except for NaClO, level L1 (application of the physical or chemical treatment) had a
332 positive effect compared to level L0 (no application of the treatment) on breaking the dormancy of
333 S. torvum seeds and improving early and final germination, as well as on the germination rate and
13
334 vigour index. In this respect, seed soaking for 12 to 24 h is known to be an efficient means for
335 improving the germination of Solanum species (Hayati et al., 2005; Ahmed et al., 2006), as it may
336 remove seed germination inhibitors (Bewley et al., 2013). Similarly, applications of the plant
337 growth regulator GA3 or KNO3 are efficient at releasing Solanum seeds from dormancy and
338 stimulating germination (Hayati et al., 2005; Wei et al., 2010; Gisbert et al., 2011a). Light
339 irradiation, which is an important regulator of seed germination in solanaceous species (Koo et al.,
340 2015), has also been observed to be efficient at stimulating germination in S. torvum seeds.
341 Amazingly, the scarification by NaClO had a highly negative effect on germination. NaClO
342 treatments are used for seed disinfection, but they also promote germination in some Solanum
343 species (Prohens et al., 1999). NaClO affects seed coat properties (Prohens et al., 1999) and this
344 may affect water uptake or other physical properties of the seed, in this case negatively, germination
345 (Bewley et al., 2013). We suggest that other suitable methods, other than NaClO treatement, for
346 seed disinfection and scarification should be used for S. torvum. Cold and heat factors also
347 influenced the germination of S. torvum such that the application of cold stratification and a heat
348 shock stimulated germination, although to a lesser extent than the other factors. In other studies,
349 cold or heat treatments proved efficient for releasing seeds of wild Solanum species from dormancy
350 (Shalimu et al., 2012; Koo et al., 2015). In this respect, cold induces the transcription GA3 synthesis
351 genes (Penfield et al., 2005), whereas heat treatments result in the production of small heat-shock
352 proteins that stimulate germination (Koo et al., 2015).
353 Seeds of S. torvum presented strong physiological dormancy and did not germinate under
354 control conditions. This strong dormancy may be the underlying reason for the poor and irregular
355 germination problems of S. torvum, thus limiting its use as a rootstock (Ginoux and Laterrot, 1991;
356 Miura et al., 1993; Gousset et al., 2005; Hayati et al., 2005), as high and rapid germination was
357 recorded with some of the treatments tested in the orthogonal array design. Although some of the
358 treatments from the orthogonal array (e.g., treatments 5 and 6) provided excellent results with high
359 levels of early and final germination as well as a high germination rate, validation of the proposed
14
360 protocol is needed. The results of the comparison of the proposed improved method with the best
361 treatment of the orthogonal array (treatment 6) confirmed the potential of the orthogonal array
362 designs to determine an optimal combination of levels for each of the factors studied (Roy, 2010).
363 The evaluation of the improved protocol under conditions that simulate commercial nursery
364 conditions (Lee et al., 2010) involved sowing the seeds in the growing substrate. Obviously, under
365 these conditions the light irradiation treatment cannot be applied as seeds were covered by soil
366 during germination. In this case, we also recorded that the control seeds of S. torvum did not
367 germinate, confirming the strong dormancy in this species (Ginoux and Laterrot, 1991; Miura et al.,
368 1993; Gousset et al., 2005; Hayati et al., 2005). However, a rapid germination in comparison to the
369 non-treated S. melongena control, was obtained with the improved germination protocol. This result
370 is important, as it indicates that this developed protocol may be applied commercially in the
371 production of S. torvum plantlets as rootstocks for eggplant grafting. The slightly lower germination
372 success compared to the Petri dish experiment may be caused by a lack of light irradiation, the
373 different germination conditions, or both (Pensfield et al., 2005; Koo et al., 2015).
374
375 5. Conclusions
376
377 Fresh S. torvum seeds present a strong dormancy exhibiting no germination. The utilization
378 of an orthogonal array design has been highly successful for estimating the main effects of factors
379 affecting the seed germination of S. torvum seeds and for establishing an improved protocol for high
380 and rapid germination. We determined that certain treatments, such as seed soaking, GA3, KNO3,
381 and light irradiation, have highly positive effects in stimulating germination, whereas NaClO
382 scarification causes negative effects. Cold scarification and heat shock also increased seed
383 germination. The improved protocol results in a high and rapid germination under Petri dish and
384 nursery growing substrate conditions. The results are of importance for the increased utilization of
385 S. torvum as a rootstock for eggplant cultivation and for breeding programmes, and these findings
15
386 also demonstrate the utility of orthogonal arrays for establishing improved protocols for seed
387 germination involving many simultaneous factors.
388
389 Acknowledgments
390
391 This work was completed as part of the initiative "Adapting Agriculture to Climate Change:
392 Collecting, Protecting and Preparing Crop Wild Relatives", which is supported by the Government
393 of Norway. The project is managed by the Global Crop Diversity Trust with the Millennium Seed
394 Bank of the Royal Botanic Gardens, Kew and is implemented in partnership with national and
395 international gene banks and plant breeding institutes. For further information see the project
396 website: http://www.cwrdiversity.org/. Isabel Andújar and Pietro Gramazio are grateful to
397 Universitat Politècnica de Valencia for their post-doctoral (PAID-10-14) and pre-doctoral
398 (Programa FPI de la UPV-Subprograma 1) contracts, respectively.
399
400 6. References
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500 Table 1
7 501 L8 orthogonal array matrix (2 ) for the seven factors evaluated (soaking, NaClO, GA3, KNO3, cold,
502 heat, and light) at two levels (L0 and L1), indicating the levels applied to each of the eight
503 treatments tested.
Factors Day of
a Treatment Soaking NaClO GA3 KNO3 Cold Heat Light initiation
1 L0 L0 L0 L0 L0 L0 L0 0
2 L0 L0 L0 L1 L1 L1 L1 -8
3 L0 L1 L1 L0 L0 L1 L1 -2
4 L0 L1 L1 L1 L1 L0 L0 -8
5 L1 L0 L1 L0 L1 L0 L1 -9
6 L1 L0 L1 L1 L0 L1 L0 -3
7 L1 L1 L0 L0 L1 L1 L0 -9
8 L1 L1 L0 L1 L0 L0 L1 -1
504 a Beginning day of the application of the different levels so that the initiation (day 0) of the
505 germination experiment is synchronized.
506
21
507 Table 2
508 Degrees of freedom, the F-ratio and its probability obtained from the ANOVA analyses for the effects of treatments and for the orthogonal
509 comparisons between the two levels for each factor tested on the Solanum torvum seed germination parameters.
Sources of Degrees of Early germination (4 d; %) Final germination (15 d; %) Germination rate (%) Vigour index
variation freedom F-ratio Prob. F F-ratio Prob. F F-ratio Prob. F F-ratio Prob. F
Treatments 7 157.5 <0.0001 238.0 <0.0001 228.8 <0.0001 235.8 <0.0001
Orthogonal contrasts
Soaking 1 202.5 <0.0001 76.9 <0.0001 149.1 <0.0001 237.9 <0.0001
NaClO 1 402.2 <0.0001 318.3 <0.0001 271.0 <0.0001 329.2 <0.0001
GA3 1 463.7 <0.0001 277.7 <0.0001 447.6 <0.0001 617.2 <0.0001
KNO3 1 9.2 0.0043 369.3 <0.0001 272.9 <0.0001 175.3 <0.0001
Cold 1 8.2 0.0066 8.2 0.0067 0.3 0.6105 0.0 0.9362
Heat 1 8.8 0.0051 54.0 <0.0001 23.5 <0.0001 12.5 0.0011
Light 1 7.8 0.0079 561.8 <0.0001 437.4 <0.0001 278.7 <0.0001
510
22
511 Table 3
512 Average values of the Solanum torvum seed germination parameters for the two levels (level 0, L0; level 1, L1) of the different factors evaluated
513 and the differences between L1 and L0 ( L1-L0).
Early germination (4 d; %) Final germination (15 d; %) Germination rate (%) Vigour index
Factors L0 L1 L1-L0a L0 L1 L1-L0 L0 L1 L1-L0 L0 L1 L1-L0
Soaking 8.2 48.2 40.0** 48.3 67.3 19.0** 42.6 65.0 22.4** 23.7 46.1 22.4**
NaClO 52.0 4.3 -47.7** 74.7 41.0 -33.7** 70.6 37.0 -33.6** 49.1 20.7 -28.4**
GA3 3.8 52.5 48.7** 42.7 73.0 30.3** 36.6 71.0 34.4** 19.1 50.7 31.6**
KNO3 26.3 30.0 3.7* 39.8 75.8 36.0** 38.5 69.2 30.7** 26.9 42.8 15.9**
Cold 26.5 29.8 3.3* 57.2 58.5 1.3* 53.7 53.9 0.2ns 34.8 34.9 0.1ns
Heat 26.0 30.3 4.3* 50.8 64.8 14.0** 48.0 59.6 11.6** 31.8 38.0 6.2*
Light 26.2 30.2 4.0* 33.5 82.2 48.7** 32.8 74.8 42.0** 23.9 45.9 22.0**
514 a**,*, ns indicate, respectively, significant at P<0.0001, P<0.01 or non-significant (see Table 2).
23
515 Table 4
516 F-ratio and its probability obtained from the ANOVA analyses for the seed germination parameters
517 resulting from the comparisons between the two treatments for Solanum torvum seed germination in
518 Petri dishes, and between the three treatments for Solanum torvum and S. melongena seed
519 germination in a commercial nursery growing substrate.
Petri dishes experimenta Commercial substrate experimentb
Parameter F-ratio Prob. F F-ratio Prob. F
Early germination 0.25 0.6309 48.26 <0.0001
Final germination 2.32 0.1586 424.37 <0.0001
Germination rate 2.06 0.1818 631.93 <0.0001
Vigour index 1.00 0.3407 551.07 <0.0001
520 a Germination treatments consisting of: (a) the optimal combination of factors of S. torvum
521 (soaking: L1; NaClO: L0; GA3: L1; KNO3: L1; cold: L1; heat: L1; light: L1) according to the
522 results obtained in the L8 orthogonal array design; (b) the best treatment of S. torvum (treatment 6;
523 soaking: L1; NaClO: L0; GA3: L1; KNO3: L1; cold: L0; heat: L1; light: L0) out of the eight tested
524 in the orthogonal array design matrix.
525 b Germination treatments consisting of: (a) the improved treatment of S. torvum consisting of the
526 optimal combination of factors (soaking: L1; NaClO: L0; GA3: L1; KNO3: L1; cold: L1; heat: L1)
527 according to the results obtained in the L8 orthogonal array design; (b) S. torvum control treatment
528 (soaking: L0; NaClO: L0; GA3: L0; KNO3: L0; cold: L0; heat: L0); (c) S. melongena control
529 treatment (soaking: L0; NaClO: L0; GA3: L0; KNO3: L0; cold: L0; heat: L0). The light factor was
530 not tested as the seeds were covered by a 7-mm layer of substrate.
24
531 Table 5
532 Average values and comparison of means for the seed emergence parameters between the three
533 treatments for Solanum torvum and S. melongena seed germination in a commercial nursery
534 growing substrate.
Treatmenta Early emergence Final emergence Emergence rate Vigour index
(7 d; %)b (14 d; %) (%)
S. torvum control 0.0 a 0.0 a 0.0 a 0.0 a
S. torvum improved 49.3 c 77.3 b 60.8 b 52.6 c
S. melongena control 14.0 b 95.3 c 63.6 b 45.9 b
a 535 S. torvum control = soaking: L0; NaClO: L0; GA3: L0; KNO3: L0; cold: L0; heat: L0. S. torvum
536 improved = soaking: L1; NaClO: L0; GA3: L1; KNO3: L1; cold: L1; heat: L1. S. melongena control
537 = soaking: L0; NaClO: L0; GA3: L0; KNO3: L0; cold: L0; heat: L0. The light factor was not tested
538 as the seeds were covered by a 7-mm layer of substrate.
539 b Means separated by different letters are significantly different according to the Student-Newman-
540 Keuls multiple range test at P<0.05.
541
25
Soaking seeds in water (1 d)
Soaking seeds in GA3 (500 ppm) (1 d)
Sowing and watering with KNO3 (1000 ppm)
Cold stratification (4ºC) (7 d)
Heat shock (37º C) (1 d)
Germination with a 16 h light (25ºC) / 8 h dark (18º C) photoperiod 542 543 Fig. 1. Schematic representation of the improved protocol for enhancing S. torvum seed
544 germination.
26
c d dd 100 A c 100 B 90 90
80 80 c (%) (%)
70 70 c 60 60 50 50 b germination germination
40 40 30 30 Final Early 20 b 20 b b 10 10 a aa a a 0 0 12345678 12345678 Treatments Treatments
ee 100 C 100 D 90 d 90 80 80 e e
(%) 70 70 c c
rate 60 60
index 50 50 d 40 b 40 Vigour c c
Germination 30 30 b 20 20 10 10 a a a a 0 0 12345678 12345678 Treatments Treatments 545 546 Fig. 2. Effect of the eight treatments tested in the L8 orthogonal array design on the four seed
547 germination parameters: (A) early germination (4 d; upper left); (B) final germination (15 d; upper
548 right); (C) germination rate (lower left); and, (D) vigour index (lower right). Bars represent the
549 standard error (SE). Means separated by different letters are significantly different according to the
550 Student-Newman-Keuls multiple range test at P<0.05.
27
100 90 80 70 (%) 60 50 40
Germination 30 20 10 0 01234567891011121314 Days 551 552 Fig. 3. Solanum torvum germination curves for two treatments: a) the improved
553 treatment consisting of the optimal combination of factors (soaking = 1; NaClO = 0;
554 GA3 = 1; KNO3 = 1; cold = 1; heat = 1; light = 1) according to the results obtained in
555 the L8 orthogonal array design results (continuous line, black circles); and, b) the best
556 treatment (treatment 6; soaking = 1; NaClO = 0; GA3 = 1; KNO3 = 1; cold = 0; heat = 1;
557 light = 0) out of the eight tested in the orthogonal array design matrix (dashed line,
558 white circles). Bars represent the standard error (SE).
559
28
100 90 80 70 (%) 60 50 40
Emergence 30 20 10 0 01234567891011121314 Days 560 561 Fig. 4. Emergence curves for Solanum torvum and S. melongena sown in a commercial
562 nursery growing substrate for three treatments: a) S. torvum control treatment (soaking
563 = 0; NaClO = 0; GA3 = 0; KNO3 = 0; cold = 0; heat = 0) (continuous line, black
564 circles); b) S. torvum improved treatment consisting of the optimal combination of
565 factors (soaking = 1; NaClO = 0; GA3 = 1; KNO3 = 1; cold = 1; heat = 1) according to
566 the results obtained in the L8 orthogonal array design (continuous line, grey circles);
567 and, c) S. melongena control treatment (soaking = 0; NaClO = 0; GA3 = 0; KNO3 = 0;
568 cold = 0; heat = 0) (dashed line, white circles). The light factor was not tested as the
569 seeds were covered by a 7-mm layer of substrate. Bars represent the standard error (SE).
29